This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cell division is a essential feature of growth, development and renewal. Each time a cell divides an exact copy of the genome must be made and each replicated chromosome equally distributed to the progeny cells. If chromosomes are unequally distributed, the cell is said to be aneuploid - the state of having the incorrect number of chromosomes. Aneuploidy is a pathological hallmark of cancerous cells and underlies a variety of birth defects. The cellular machinery to prevent aneuploidy is called the mitotic checkpoint and acts to prevent distribution of the chromosomes until they are attached to the mitotic spindle. The target of the the checkpoint activity is an E3 Ubiquitin Ligase called the Anaphase-promoting complex (APC/C). Current models of checkpoint function revolve around protein complexes produced by unattached kinetochores that directly inhibit the activity of the APC/C. The nature, i.e. composition and post-translational state, of these complexes have remained difficult to study. Through the use of tandem affinity methods, we have purified complexes for many of the checkpoint proteins from cells arrested in mitosis. From such cells we expect to identify complexes that represent one or more species generated by unattached chromosomes. In parallel, we are also carrying out live cell microscopic analyses using fluorescence correlation methods to follow these complexes in living cells. Permitting the analysis of the complex of interest both in vitro by mass spectrometry and in vivo by fluorescence methods.